Bottom Line:
Similarly, 566 transcripts are differentially expressed when CCs mature with (CCs+OO) or without (CCs-OO) their enclosed oocytes.Similarly, while transcripts over expressed in OO+CCs are involved in carbohydrate metabolism (ACO1, 2), molecular transport (GAPDH, GFPT1) and nucleic acid metabolism (CBS, NOS2), those over expressed in CCs+ OO are involved in cellular growth and proliferation (FOS, GADD45A), cell cycle (HAS2, VEGFA), cellular development (AMD1, AURKA, DPP4) and gene expression (FOSB, TGFB2).In conclusion, this study has generated large scale gene expression data from different oocyte and CCs samples that would provide insights into gene functions and interactions within and across different pathways that are involved in the maturation of bovine oocytes.

Background: The bi-directional communication between the oocyte and its companion cumulus cells (CCs) is crucial for development and functions of both cell types. Transcripts that are exclusively expressed either in oocytes or CCs and molecular mechanisms affected due to removal of the communication axis between the two cell types is not investigated at a larger scale. The main objectives of this study were: 1. To identify transcripts exclusively expressed either in oocyte or CCs and 2. To identify those which are differentially expressed when the oocyte is cultured with or without its companion CCs and vice versa.

Results: We analyzed transcriptome profile of different oocyte and CC samples using Affymetrix GeneChip Bovine Genome array containing 23000 transcripts. Out of 13162 genes detected in germinal vesicle (GV) oocytes and their companion CCs, 1516 and 2727 are exclusively expressed in oocytes and CCs, respectively, while 8919 are expressed in both. Similarly, of 13602 genes detected in metaphase II (MII) oocytes and CCs, 1423 and 3100 are exclusively expressed in oocytes and CCs, respectively, while 9079 are expressed in both. A total of 265 transcripts are differentially expressed between oocytes cultured with (OO+CCs) and without (OO-CCs) CCs, of which 217 and 48 are over expressed in the former and the later groups, respectively. Similarly, 566 transcripts are differentially expressed when CCs mature with (CCs+OO) or without (CCs-OO) their enclosed oocytes. Of these, 320 and 246 are over expressed in CCs+OO and CCs-OO, respectively.While oocyte specific transcripts include those involved in transcription (IRF6, POU5F1, MYF5, MED18), translation (EIF2AK1, EIF4ENIF1) and CCs specific ones include those involved in carbohydrate metabolism (HYAL1, PFKL, PYGL, MPI), protein metabolic processes (IHH, APOA1, PLOD1), steroid biosynthetic process (APOA1, CYP11A1, HSD3B1, HSD3B7). Similarly, while transcripts over expressed in OO+CCs are involved in carbohydrate metabolism (ACO1, 2), molecular transport (GAPDH, GFPT1) and nucleic acid metabolism (CBS, NOS2), those over expressed in CCs+ OO are involved in cellular growth and proliferation (FOS, GADD45A), cell cycle (HAS2, VEGFA), cellular development (AMD1, AURKA, DPP4) and gene expression (FOSB, TGFB2).

Conclusion: In conclusion, this study has generated large scale gene expression data from different oocyte and CCs samples that would provide insights into gene functions and interactions within and across different pathways that are involved in the maturation of bovine oocytes. Moreover, the presence or absence of oocyte and CC factors during bovine oocyte maturation can have a profound effect on transcript abundance of each cell types, thereby showing the prevailing molecular cross-talk between oocytes and their corresponding CCs.

Figure 20: Validation of the microarray data by semi quantitative RT-PCR. A 2% agarose gel electrophoresis depicting the mRNA expression of genes that are exclusively expressed either in oocytes or CCs. Number 1 shows a negative control (dd water as a template) and 2, 3, 4 and 5 show the abundance levels of each transcript in MII CCs, MII oocytes, GV CCs and GV oocytes, respectively. 18S was used as a loading control for total RNA.

Mentions:
In order to validate the micro array data, a total of 23 transcripts were quantified using qRT-PCR as described in materials and methods section. The assayed genes are selected based on the criteria of being abundant at significantly higher level or their exclusive abundance in either of the samples considered in the array analysis. Transcripts Semi-quantitative RT-PCR was also adopted to validate some genes that are exclusively expressed either as the oocyte or CC transcripts. With the exception of one transcript, the results of qRT-PCR analysis validated the array data as the expression levels of a given transcript between the two samples compared (designated by *) are significantly different (P < 0.05) (Figures 15, 16, 17, 18, 19). Additionally, semi-quantitative RT-PCR validation of some selected genes also supports the microarray data (Figure 20). The actual microarray expression values of genes validated using real time qRT-PCR analysis are shown as additional files 20, 21, 22, 23 and 24.

Figure 20: Validation of the microarray data by semi quantitative RT-PCR. A 2% agarose gel electrophoresis depicting the mRNA expression of genes that are exclusively expressed either in oocytes or CCs. Number 1 shows a negative control (dd water as a template) and 2, 3, 4 and 5 show the abundance levels of each transcript in MII CCs, MII oocytes, GV CCs and GV oocytes, respectively. 18S was used as a loading control for total RNA.

Mentions:
In order to validate the micro array data, a total of 23 transcripts were quantified using qRT-PCR as described in materials and methods section. The assayed genes are selected based on the criteria of being abundant at significantly higher level or their exclusive abundance in either of the samples considered in the array analysis. Transcripts Semi-quantitative RT-PCR was also adopted to validate some genes that are exclusively expressed either as the oocyte or CC transcripts. With the exception of one transcript, the results of qRT-PCR analysis validated the array data as the expression levels of a given transcript between the two samples compared (designated by *) are significantly different (P < 0.05) (Figures 15, 16, 17, 18, 19). Additionally, semi-quantitative RT-PCR validation of some selected genes also supports the microarray data (Figure 20). The actual microarray expression values of genes validated using real time qRT-PCR analysis are shown as additional files 20, 21, 22, 23 and 24.

Bottom Line:
Similarly, 566 transcripts are differentially expressed when CCs mature with (CCs+OO) or without (CCs-OO) their enclosed oocytes.Similarly, while transcripts over expressed in OO+CCs are involved in carbohydrate metabolism (ACO1, 2), molecular transport (GAPDH, GFPT1) and nucleic acid metabolism (CBS, NOS2), those over expressed in CCs+ OO are involved in cellular growth and proliferation (FOS, GADD45A), cell cycle (HAS2, VEGFA), cellular development (AMD1, AURKA, DPP4) and gene expression (FOSB, TGFB2).In conclusion, this study has generated large scale gene expression data from different oocyte and CCs samples that would provide insights into gene functions and interactions within and across different pathways that are involved in the maturation of bovine oocytes.

Background: The bi-directional communication between the oocyte and its companion cumulus cells (CCs) is crucial for development and functions of both cell types. Transcripts that are exclusively expressed either in oocytes or CCs and molecular mechanisms affected due to removal of the communication axis between the two cell types is not investigated at a larger scale. The main objectives of this study were: 1. To identify transcripts exclusively expressed either in oocyte or CCs and 2. To identify those which are differentially expressed when the oocyte is cultured with or without its companion CCs and vice versa.

Results: We analyzed transcriptome profile of different oocyte and CC samples using Affymetrix GeneChip Bovine Genome array containing 23000 transcripts. Out of 13162 genes detected in germinal vesicle (GV) oocytes and their companion CCs, 1516 and 2727 are exclusively expressed in oocytes and CCs, respectively, while 8919 are expressed in both. Similarly, of 13602 genes detected in metaphase II (MII) oocytes and CCs, 1423 and 3100 are exclusively expressed in oocytes and CCs, respectively, while 9079 are expressed in both. A total of 265 transcripts are differentially expressed between oocytes cultured with (OO+CCs) and without (OO-CCs) CCs, of which 217 and 48 are over expressed in the former and the later groups, respectively. Similarly, 566 transcripts are differentially expressed when CCs mature with (CCs+OO) or without (CCs-OO) their enclosed oocytes. Of these, 320 and 246 are over expressed in CCs+OO and CCs-OO, respectively.While oocyte specific transcripts include those involved in transcription (IRF6, POU5F1, MYF5, MED18), translation (EIF2AK1, EIF4ENIF1) and CCs specific ones include those involved in carbohydrate metabolism (HYAL1, PFKL, PYGL, MPI), protein metabolic processes (IHH, APOA1, PLOD1), steroid biosynthetic process (APOA1, CYP11A1, HSD3B1, HSD3B7). Similarly, while transcripts over expressed in OO+CCs are involved in carbohydrate metabolism (ACO1, 2), molecular transport (GAPDH, GFPT1) and nucleic acid metabolism (CBS, NOS2), those over expressed in CCs+ OO are involved in cellular growth and proliferation (FOS, GADD45A), cell cycle (HAS2, VEGFA), cellular development (AMD1, AURKA, DPP4) and gene expression (FOSB, TGFB2).

Conclusion: In conclusion, this study has generated large scale gene expression data from different oocyte and CCs samples that would provide insights into gene functions and interactions within and across different pathways that are involved in the maturation of bovine oocytes. Moreover, the presence or absence of oocyte and CC factors during bovine oocyte maturation can have a profound effect on transcript abundance of each cell types, thereby showing the prevailing molecular cross-talk between oocytes and their corresponding CCs.